Genome-wide investigation of GRAM-domain containing genes in rice reveals their role in plant-rhizobacteria interactions and abiotic stress responses
Introduction
Rice (Oryza sativa L.) is a widely consumed staple cereal grain across the globe, especially in Asia. Due to various abiotic stresses such as salt, drought, cold, as well as biotic stresses, rice losses their tolerance capability that results in declination of crop productivity worldwide. In the present scenario, abiotic stresses showed an adverse impact on rice production by decreasing the yields up to 70% [1]. It is considered that the early seedling stage of rice is comparatively more sensitive to stress than at the reproductive stage [2,3]. In rice, abiotic stress adversely affects all major metabolic activities such as accumulation of compatible solutes including proline, and soluble sugars, reduction in photosynthesis, cell wall damage, and an overall decline in germination and seedling growth [4]. The application of plant growth-promoting rhizobacteria (PGPR) is recently gaining global acceptance as an alternate technology to ameliorate abiotic stress in plants [2,[5], [6], [7]]. Bacillus amyloliquefaciens strain SN13, a PGPR has been reported to improve salt stress tolerance of rice seedlings [2,8]. A transcriptome study of rice roots inoculated with SN13 under salt stress revealed significant upregulation of a GRAM-domain containing gene [9]. However, the molecular mechanism of SN13-mediated salt stress tolerance involving GRAM-domain containing genes is yet to be unravelled.
The GRAM domain is highly conserved and is known to be found in glucosyltransferases, myotubularin, as well as in other membrane-associated proteins. This region was named GRAM domain after the better characterization of glucosyltransferases, Rab-like GTPase activators and myotubularin [10]. Generally, it consists of ~70 aa residues and predicted to form four β-strand and one α-helix. This domain is expected to be involved signaling processes that are membrane-associated, i.e. intracellular protein or lipid-bound signaling pathways. Their roles are widely studied in the binding of animals myotubularin proteins and phosphoinositol. Among plants, the GRAM-domain family members appear responsive to ABA and involved in the perception and regulation of environmental and hormonal signaling under various stress conditions [[11], [12], [13], [14]]. Based on sequence homology with Hordeum vulgare, the first isolated gene that contains GRAM domain sequence was aba45, identified in plants that mainly expressed in developing aleurone and was upregulated by ABA [15]. Additionally, VAD1 was found to encode a GRAM domain that is salicylic acid (SA) dependent and is required for the mechanism of programmed cell death resulting, enhanced disease resistance to bacterial pathogens [16]. Among these, few proteins contain only GRAM domain; while other remaining contain conserved domains like Beach, C2 domain, TBC, WD40, and so on along with GRAM domain. Among all these domains, C2-GRAM domain-containing proteins might be only found in plants [17]. Many GRAM domain proteins harbor additional lipid-binding motifs, e.g., FVVE and pleckstrin domain (PH domain) which are thought to be associated with intracellular membranes.
Jiang et al. [14] studied the transcriptional profiling of GRAM domain family in different eukaryotes representing plants, animals, and yeast. In O. sativa and Arabidopsis Jiang et al. [14] identified 17 and 13 GRAM domain-containing gene respectively. Although, a few GRAM domain family members have been identified in rice and Arabidopsis, and some of them were found to be involved in various abiotic and biotic stress responses, however, no data is available so far illustrating the role of any PGPR on gene expression modulation of all the domain members and their involvement under various abiotic stresses and phytohormones in rice. Furthermore, in this gene family, the evolutionary analysis was also not done. Therefore, the present study reported the genome-wide identification of the GRAM-domain family in rice and their expression analysis in rice roots exposed to salinity in the presence of SN13. Their genomic distribution, phylogenetic analysis, functional annotation, domain combinations, gene duplications, and substitution rates were also analyzed to clearly explain their evolution. We also surveyed their expression patterns under different abiotic stresses with and without PGPR based on our experimental data and publically available expression data at cellular and tissue levels to predict their functions and role in stress tolerance and beneficial plant-microbe interactions.
Section snippets
Identification of GRAM domain in rice
The Hidden Markov Model (HMM) profile of the GRAM domain (PF02893) was prepared from Pfam v27.0 database (http://pfam.xfam.org/) and queried against the PHYTOZOME database of Oryza sativa (www.phytozome.jgi.doe.gov/). All hits with E < 1.0 were selected, and the presence of conserved GRAM domain was confirmed through HMMSCAN (http://hmmer.janelia.org/search/hmmscan).
Chromosomal location and gene structure of GRAM domain
The GRAM domain-containing proteins were queried against O. sativa genome using BLASTP with default settings, and the physical map
Identification of the GRAM domain-containing protein in the Oryza sativa genome
The HMM BLAST detected a total of 94 GRAM domain-containing protein sequences from Oryza sativa. After removal of splice variants (30 proteins) of primary transcripts, a total of 64 putative GRAM domain-containing proteins (Supporting information S2) remained. Gene LOC_Os05g30750 was having maximum number of alternate transcripts (six) followed by LOC_Os02g10480 and LOC_Os07g22640 with 5 and 3 splice variants, respectively (Supporting information S2). Genes LOC_Os01g62430, LOC_Os06g40704,
Conclusion
The GRAM domain family genes play as an important regulator for plant growth and development as well as stress responses, and hence the present study characterizes the rice GRAM domain family genes using in silico tools and also analyze their expression level under abiotic stress and phytohormone treatments under the influence of SN13. In this study total 64 GRAM domain-containing proteins were identified in rice that was ~4 fold higher in number than reported by Jiang et al. [14]. Further
CRediT authorship contribution statement
Shalini Tiwari: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing - original draft, Writing - review & editing. Shweta Shweta: Data curation, Methodology, Formal analysis, Software, Validation. Manoj Prasad: Supervision, Visualization, Writing - review & editing. Charu Lata: .
Acknowledgments
CL acknowledges “Early Career Research Award (ECRA)” by Science and Engineering Research Board (SERB), Government of India [Grant No. ECR/2017/001593]. Authors are highly thankful to Dr. Puneet Singh Chauhan, CSIR-National Botanical Research Institute, Lucknow, India for providing the bacterial strain and other required facilities for the study.
Declaration of competing interest
Authors declare no conflict of interest.
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